Shigella flexneri is a gram-negative enteric pathogen that causes bacillary dysentery (shigellosis), which continues to be an important worldwide public health problem. An essential step in the pathogenesis of shigellosis is bacterial invasion of the epithelial ceils that line the colon. Invasion plasmid antigen C (IpaC) is the major effector protein that subverts normal epithelial cell signaling to promote Shigella uptake. Our lab and others have made a number of advances in understanding the structure-function relationship of IpaC; however, there has been little focus thus far on determining the precise protein chemistry of IpaC-directed epithelial cell invasion. This is partly due to difficulties in purifying and handling this protein. The long-range goal of our laboratory is to determine the precise functional and structural organization of IpaC and to elucidate the molecular and structural basis for IpaC interaction with and subversion of epithelial cell signaling molecules. The specific aims of this project are: 1) to determine the sequence, structural and physical features present in the IpaC N-terminal domain that are involved in its translocation to the host cell membrane; 2) to determine the molecular basis for the role of the IpaC C-terminus in directing vacuolar escape of internalized Shigella and subverting the host cell signals that control the host cell cytoskeleton; and 3) to generate crystals (or co-crystals) of IpaC and IpaC fragments for high-resolution structure analysis. Completion of this work will reveal important underlying principles of the mechanism by which Shigella promotes an epithelial cell to become phagocytic for the benefit of the pathogen. This will also allow us identify potential targets for chemotherapeutic control of infection by Shigella and related human pathogens. Lastly, by determining the biochemical and structural basis for IpaC function, it may be possible to devise ways to use this protein for beneficial purposes since this protein is not toxic to mammalian cells but it may have the ability to present antigens to the mucosal immune system.